Cephalosporin-NO-donor prodrug PYRRO-C3D shows β-lactam-mediated activity against Streptococcus pneumoniae biofilms

Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3\u27-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with β-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following β-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (β-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks β-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of β-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the β-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections

Low concentrations of nitric oxide modulate Streptococcus pneumoniae biofilm metabolism and antibiotic tolerance

Streptococcus pneumoniae is one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence of S. pneumoniae in chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesised that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO, did not result in S. pneumoniae biofilm dispersal. Instead, treatment of both in vitro biofilms and ex vivo adenoid tissue samples (a reservoir for S. pneumoniae biofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO therefore appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms

Mast cell chemoattractants in allergic rhinitis

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mast cell chemoattractants in allergic rhinitis

Epithelial mast cell accumulation is a characteristic feature of allergic rhinitis.  The mechanism for this tissue localization is undefined.  This study has evaluated the nasal expression of the known mast cell chemoattractants eotaxin, stem cell factor (SCF) and the transforming growth factor-beta (TGF-β) isoforms, TGF-β1, TGF-β2 and TGF-β3, along with their receptors CCR-3, c-kit, and TGF-βRI, -βRII, -βRIII, respectively.  The initial studies focused on immunohistochemical expression of protein in nasal biopsy specimens from rhinitic (perennial and seasonal allergic rhinitis (SAR)) and non-rhinitic subjects (healthy controls and asymptomatic out of season, seasonal rhinitis), evaluating the level of expression and tissue localization.  Based on these results, the study extended to the evaluation of nasal lavage measures of luminal protein in the same study groups for TGF-β1, TGF-β2 and eotaxin.  These measures were related to symptom reporting and lavage alpha 2-macroglobulin (α2MG), as indices of clinical disease severity and nasal airway inflammation respectively, and more specifically to intra-luminal eosinophil accumulation, as represented by cytospin differential cell count.  Subsequently, evaluation was undertaken of TGF-β1, -β2 and –β3, TGF-β RI, - β RII and – β RIII and eotaxin gene expression by real-time quantitative polymerase chain reaction (TaqMan RT-PCR) in whole biopsies, and compared to that for tumour necrosis factor-alpha (TNF-α), as a positive control, and connective tissue growth factor (CTGF), as a downstream marker of TGF-β bioactivity, in the same populations.  This study was extended to also evaluate gene expression for Smad proteins.  Finally to understand the basis for TGF-β expression in epithelial cells, a series of in vitro studies, involving evaluation of both protein release into supernatant fluid and tissue gene expression, were undertaken on RPMI 2650 cells, a nasal epithelial cell line, investigating the effects of allergens (house dust mite (HDM) and grass pollen (GP)), TNF-α and the Th2 cytokines interleukin (IL)-4 and IL-13. Evidence was found of significantly increased epithelial immunoreactivity for TGF-β1, -β2, -βRII, and – βRIII in the perennial and seasonal allergic rhinitis compared with healthy controls.  TGF-βRI and –β RII were found to co-localise to mast cells.  There was no increase in SCF immunoreactivity within the epithelium in naturally occurring disease.  Significant correlations were found between epithelial immunoreactivity for TGF-β1, -β2, -βRI, -βRII and the number of epithelial mast cells.  These findings of enhanced epithelial TGF-β immunoreactivity in allergic rhinitis, the correlation with intra-epithelial mast cell numbers, and the co-localisation of TGF-β receptors to mast cells, suggest that the epithelial expression of TGF-β may represent an important biological process involved in either the recruitment or retention of mast cells within the epithelium in naturally occurring allergic rhinitis.</p

Dataset supporting the University of Southampton Doctoral Thesis &quot;Strain-specific Intracellular Staphylococcus aureus in resistant chronic rhinosinusitis. Disease mechanisms and potential novel therapies&quot;Increased carriage of virulence genes may mediate enhanced pathogenicity of chronic rhinosinusitis-related S. aureus strains sequence data

Dataset supporting the University of Southampton Doctoral Thesis &quot;Strain-specific Intracellular Staphylococcus aureus in resistant chronic rhinosinusitis. Disease mechanisms and potential novel therapies&quot; The data presents raw results of experiments that display increased carriage of virulence genes that may mediate enhanced pathogenicity of chronic rhinosinusitis-related S. aureus strains sequence data. The data focuses on paired sequencing reads of Staphylococcus aureus cultures and any supplementary data that was used in &quot;Strain-specific Intracellular Staphylococcus aureus in resistant chronic rhinosinusitis. Disease mechanisms and potential novel therapies&quot; 4 files with S.aureus sequences cultured from control subjects (control_strains.zip), CRSsNP subjects (CRSsNP_strains.zip) and CRSwNP subjects (CRSwNP_strains.zip) and all remaining PhD data (PhD_data_zip.zip).</span